Medicago LINC Complexes Function in Nuclear Morphology, Nuclear Movement, and Root ... · dopsis root hairs (Tamura et al., 2013; Zhou et al., 2015a). Further, WIP, WIT, and Myosin

Medicago LINC Complexes Function in NuclearMorphology, Nuclear Movement, and RootNodule Symbiosis1[OPEN]

Anna H. Newman-Griffis,a,b Pablo del Cerro,c,2 Myriam Charpentier,c,3 and Iris Meiera,b,3,4

aDepartment of Molecular Genetics, The Ohio State University, Columbus, OhiobCenter for RNA Biology, The Ohio State University, Columbus, OhiocCell and Developmental Biology, John Innes Centre, Norwich, UK

ORCID IDs: 0000-0001-5490-234X (A.H.N.); 0000-0002-8030-7226 (P.d.C.); 0000-0003-3784-3039 (M.C.); 0000-0002-4141-5400 (I.M.).

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker ofNucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes areinvolved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targetednuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been establishedwhether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legumeMedicago truncatula. We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUNprotein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUNdominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movementin Medicago root hairs, and are important for infection thread initiation and nodulation.

Mechanisms of nuclear dynamics have been charac-terized in both plants and opisthokonts, in whichnuclear movement and positioning are vital for de-velopmental processes such as Caenorhabditis eleganshypodermal cell development and Drosophila mela-nogaster eye disk development (for review, see Starr andFridolfsson, 2010). Nuclear movement is orchestratedin conjunction with the cytoskeleton, often through at-tachment to nuclear envelope (NE)-bridging linker of

nucleoskeleton and cytoskeleton (LINC) complexes(Crisp et al., 2006). LINC complexes consist of innernuclear membrane Sad1/UNC-84 (SUN) proteins andouter nuclear membrane (ONM) Klarsicht/ANC-1/Syne Homology (KASH) proteins. SUN and KASHproteins interact in the NE lumen through an interac-tion of the terminal four amino acids of the KASHproteins (here defined as the SUN-interacting tail, orSIT, domain) with the SUNdomain of the SUN proteins(Starr and Fridolfsson, 2010; Zhou et al., 2014). AsKASH proteins interact with cytoskeletal componentsand motor proteins, and SUN proteins interact withnuclear lamins and chromatin-associated proteins, LINCcomplexes connect the cytoskeleton to the nuclearinterior (Rothballer and Kutay, 2013; Luxton and Starr,2014).In plants, nuclear movement is associated with pro-

cesses ranging from blue-light avoidance to the ac-commodation of arbuscular mycorrhizal fungi (Griffiset al., 2014). Plant genomes encode SUN proteins, butno proteins with sequence similarity to animal KASHproteins have been discovered (Graumann et al., 2010;Oda and Fukuda, 2011). Recently, it was established inArabidopsis (Arabidopsis thaliana) that ONM-localizedtryptophan-proline-proline (WPP) domain-interactingproteins (WIPs) are plant analogs of animal KASHproteins, binding SUN proteins at the NE (Zhou et al.,2012a). The WIPs and their binding partners, the WPP-interacting tail-anchored proteins (WIT1 andWIT2), areinvolved in RanGAP anchoring to the NE (Xu et al.,2007; Zhao et al., 2008) and nuclear movement in root

1This work was supported by funding from the National ScienceFoundation (grants no. NSF-1440019 and no. NSF-1613501), an OhioState Center for RNA Biology fellowship (to A.H.N.-G.), the Biotech-nology and Biological Sciences Research Council (BBSRC grant no.BB/P007112/1 to M.C.), and the European Molecular Biology Orga-nization (EMBO short-term fellowship no. 6995 and Spanish studentfellowship no. FPU14-00160 to P.d.C.).

2Present address: Department of Microbiology, Seville University,Seville, Spain.

3Senior authors.4Author for contact: [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is: IrisMeier ([email protected]).

A.H.N.-G. and I.M. conceived and planned the experiments; I.M.and M.C. supervised the experiments; A.H.N.-G. performed and an-alyzed most of the experiments and wrote the article with the contri-bution of all authors; P.d.C. performed and analyzed infection-threadexperiments.

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hairs (Zhou et al., 2012a; Tamura et al., 2013; Zhou andMeier, 2013) and pollen tubes (Zhou and Meier, 2014).

Animal KASH proteins have a common signature, ashort C-terminal domain consisting of a transmem-brane domain (TMD), a linker of ;40 amino acids, anda terminal 4-amino acid motif with the consensus se-quence PPPT (Starr and Fridolfsson, 2010). Based onWIP domain organization, as well as metazoan KASHproteins, a plant KASH protein signature was defined.Similar to metazoan KASH, this signature contains aC-terminal TMD, a short linker, and a terminal four-amino acid motif (i.e., SUN-interacting tail [SIT] do-main). However, the linker is shorter, and the terminalfour-amino acid motif follows a [DTVAMPLIFY][VAPIL]PT pattern (Zhou et al., 2014). Based on this signa-ture, 10 more potential plant KASH proteins wereidentified in Arabidopsis that have no sequence simi-larity to animal KASH proteins, and five candidateswere shown to be NE-localized, bind SUN proteins,and require SUN for NE location, the hallmarks ofKASHproteins (Zhou et al., 2014). Of those, ArabidopsisSINE1 is involved in actin-dependent nuclear posi-tioning in guard cells, and its paralog SINE2 contributesto innate immunity against an oomycete pathogen(Zhou et al., 2014).

In root hairs, nuclei maintain a consistent distancefrom the tip during development, 77 6 15 mm in Ara-bidopsis and 30 6 5 mm in Medicago (Medicago trunca-tula; Sieberer and Emons, 2000; Ketelaar et al., 2002).The nucleus is connected to the tip of the root hair byactin in Arabidopsis and microtubules (MTs) in le-gumes, but subsequent random nuclear movement af-ter growth termination is actin-dependent in bothcontexts (Lloyd et al., 1987; Ketelaar et al., 2002; Siebereret al., 2002). Nuclei in root hairs, as well as in manyother differentiated plant cells, are elongated and oftenspindle-shaped. Over multiple studies, it has beenshown that a LINC complex composed of SUN, WIP,WIT, and the motor protein Myosin XI-i is the primarymechanism driving elongated nuclear shape in Arabi-dopsis root hairs (Tamura et al., 2013; Zhou et al.,2015a). Further, WIP, WIT, and Myosin XI-i havebeen shown to be necessary for the random nuclearmovements associated with mature root hairs. How-ever, the role of SUN proteins in nuclear movement inroot hairs has not been characterized, and the functionof LINC complexes involving a KASH protein otherthan WIP in root hairs is not known.

Root nodule symbiosis between legumes andnitrogen-fixing rhizobia requires the coordinated de-velopment of two new structures, the infection thread(IT) from the root hair cell and the root nodule fromthe underlying root cortex (reviewed in Oldroyd andDownie, 2008). During root hair elongation, the nucleusmaintains a constant distance from the tip, then movesto a random location once development ceases (Siebererand Emons, 2000; Ketelaar et al., 2002). In responseto lipochitooligosaccharides (the so-called nodula-tion factors) secreted by rhizobia, root hair tips swelland reinitiate growth. The root hair nucleus moves to a

location near the root hair tip, which curls around andtraps the rhizobia to form an infection pocket (Siebererand Emons, 2000; Gage, 2004). The plasma membraneinvaginates, forming the IT, a tubular structure towhich the nucleus is connected by dense MTs(Fåhraeus, 1957; Timmers et al., 1999). Subsequent ITprogression within the root hair follows the path of themoving nucleus, regardless of its direction, supportingthe idea that nuclear movement is necessary for ITguidance (Fåhraeus, 1957). Meanwhile, in the under-lying cortical cells, the nucleus moves from the cellperiphery to the center, and a bridge of cytoplasmic andcytoskeletal elements called the “pre-infection thread”forms around it (Bakhuizen, 1988; van Brussel et al.,1992), paving the way for IT progression. Based onthe intimate association of nuclear movement and po-sitioning in several stages of rhizobial symbiosis, it hasbeen suggested, but not yet established, that nuclearmovement plays a role in nodulation initiation(Fåhraeus, 1957; Suzaki et al., 2015).

In this study, we translate the work on ArabidopsisLINC complexes to the model legume M. truncatula,with the ultimate goal of testing whether the movementof the nucleus is essential to rhizobial infection. Webioinformatically identified nine Medicago KASH pro-teins, two ONM-KASH–interacting proteins (WITs),and one C-terminal SUN protein encoded by the Med-icago genome. These LINC complex protein candidateswere verified by diagnostic protein–protein interac-tions and in vivo localization patterns. We furtherdemonstrate that disrupting KASH protein preferentialassociation with the NE leads to alterations in root hairnuclear morphology and mobility, as well as defects inrhizobial infection. Our data indicate that LINC com-plexes play a role in the dynamic interaction ofMedicagoroot hair nuclei with their cellular environment, which,in turn, influences symbiosis initiation.

RESULTS

The Medicago Genome Encodes Putative LINCComplex Components

To identify putative LINC complex componentsencoded by the Medicago genome, we employed boththe basic local alignment search tool (BLASTp) and the“Does It Robustly” (DORY) algorithm (Zhou et al.,2014). Searching the translated Medicago genome se-quence (version 4.0) with the amino acid sequences ofknown Arabidopsis LINC complex components usingBLASTp, we identified putative homologs of Arabi-dopsis KASH proteins WIP1 (MtWIP1a, MtWIP1b) andSINE1 (MtSINE1), a putative homolog of ArabidopsisSUN1 and SUN2 (MtSUN), as well as two putativehomologs of the WIP-interacting outer NE proteinsWIT1 and WIT2 (MtWIT1 and MtWIT2 (Fig. 1A;Supplemental Table S1). MtSINE5a and MtSINE5bwere previously identified (Zhou et al., 2014). We usedthe DORY algorithm (Zhou et al., 2014) to identify

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additional putative KASH proteins not conserved be-tween Medicago and Arabidopsis. DORY searches for apredicted TMD followed by a variable linker of amaximum of 36 amino acids and the four-amino acidterminal motif [DTVAMPLIFY][VAIPL]PT. DORYidentified not only the KASH proteins that were con-served from Arabidopsis, but also four additionalMedicago open reading frames (ORFs) encoding pro-teins with C-terminal KASH domain signatures,which were termed KASH1, KASH4, KASH5, andKASH6 (Fig. 1B; Supplemental Table S1). Of those,KASH4 and KASH5 are putative paralogs. KASH1,KASH4, KASH5, and KASH6 do not contain anyknown functional domains. Whereas KASH6 appearslimited to the Fabaceae, KASH1, KASH4, and KASH5are conserved throughout the Rosids, but excludedfrom the Fagales, Cucurbitales, Celastrales, Cross-omatales, Myrtales, Geraniales, and, crucially, theBrassicales (Fig. 1, C and D; Supplemental Table S2;Alignment and color are ClustalX; Jeanmougin et al.,1998), explaining why they were not previouslyidentified in Arabidopsis.To gain insight into potential functions of these pu-

tative LINC complex components, we compared theirexpression level based on publicly available Affymetrixdata (Benedito et al., 2008; He et al., 2009; probes usedare shown in Supplemental Table S1). Putative Medi-cago LINC complex components have a variety of ex-pression levels in different M. truncatula tissues(Supplemental Fig. S1). MtWIP1b is expressed at severalorders of magnitude higher levels thanMtWIP1a acrossall tissues analyzed. Interestingly, MtWIP1a is almostexclusively expressed in arbusculated cells of theroot cortex. MtWIP1b and MtWIT1 are coexpressed,but MtWIP1b is expressed at much higher levels thanMtWIT1. MtSUN is expressed at levels comparable toMtWIP1b, and is relatively ubiquitous. MtSINE1 isexpressed throughout the plant, with peak expressionlevels in seeds, pods, vegetative buds and shoots, roottips, and stem internodes, but also including root hairsand arbusculated cells at lower levels. These may sug-gest a role for MtSINE1 in cycling cells. MtSINE5a isalso relatively evenly expressed, but at a much lowerlevel than other KASH proteins. There are currently noprobe sets for MtSINE5b, MtKASH1, MtKASH4,MtKASH5, andMtKASH6. Together, these data suggestthat LINC complexes are expressed and may functionin a wide range of Medicago tissues, including roots.

Putative Medicago LINC Complex Components AreLocalized to the NE in a SIT-Dependent Manner inNicotiana benthamiana

Because of their limited sequence similarity, KASHproteins have been defined by their localization at theNE and physical interaction with SUN proteins. BothNE localization and SUN protein binding depend onthe C-terminal SIT domain of KASH proteins and astretch of amino acids within the SUN domain called

Figure 1. In silico analysis of putative Medicago KASH proteins. A,Protein domain organization of putative KASH proteins. (Yellow)coiled-coil; (red) armadillo repeat; (purple) SUN domain; (green) TMDhelix; (blue) unknown; (numbers) amino acids. For all KASH proteins,the terminal four amino acids are shown in single-letter code. B to D,ClustalX alignment of KASH proteins’ TMD domains and SITs for (B)confirmedMedicago KASH proteins, (C) MtKASH1 and homologs, and(D) putatively paralogous proteins MtKASH4 and MtKASH5 and theirhomologs. (Color) ClustalX amino acid groups (Jeanmougin et al.,1998); (right) total number of amino acids. Homologs of KASH1 andKASH4/KASH5 in (C) and (D) are listed by their organism of origin,given numbers in the case of multiple protein sequences per organism,and were identified using BLASTp. Full GenBank accession numbersare provided in Supplemental Table S1.

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the “KASH-binding lid” (Sosa et al., 2012; Zhou et al.,2012b).

We acquired the ORFs of all putative LINC complexcomponents shown in Figure 1A, either by amplify-ing cDNA from Medicago ecotype R108 seedlings(MtWIP1a, MtWIP1b, MtWIT2, MtSINE5a, MtSINE1,MtKASH1, MtKASH4, MtKASH5) or by commercialsynthesis based on theA17 genome (GenScript;MtWIT1,MtSINE5b, MtKASH6, MtSUN). The subcellular locali-zation of 35S promoter-driven green-fluorescent-protein (GFP)–fusion proteins was determined bytransient expression in N. benthamiana leaf epidermalcells via Agrobacterium tumefaciens–mediated leaf infil-tration. Themajority of the KASHprotein candidates arelocated at the NE in N. benthamiana (Fig. 2A). MtSUN isassociatedwith the NE andmutating the KASH-bindinglid (MtSUNdmut) does not alter this localization (Fig. 2Band see below). Finally, the proposedMtWIP interactionpartner MtWIT1 is also associated with the NE (Fig. 2B).Two candidates from the bioinformatic screen werenegative for NE localization—MtWIT2 and MtKASH6(Fig. 2C).We consider these false positives of the originalselection criteria.

To determine if NE localization of the KASHprotein candidates depends on the four-amino acid SIT

domain, deletion constructs lacking the SIT domain(KASHDSIT) were tested. This mutation was previ-ously shown to significantly reduce the NE targetingand SUN protein binding of Arabidopsis KASH pro-teins (Zhou et al., 2014). NE enrichment was signifi-cantly reduced for the Medicago KASHΔSIT proteinsrelative towild-type versions (Fig. 2, A andD, Student’st test P , 0.00001). This suggests that retention by in-teraction with SUN is required for the NE accumulationof these KASH proteins, as was shown previously forboth plant and animal KASH proteins (Starr andFridolfsson, 2010; Zhou et al., 2012a).

To determine whether these putative LINC complexcomponents localize to the NE in M. truncatula roots,GFP fusions of the NE-localized LINC complex candi-dates were expressed in transgenic hairy roots of Med-icago R108 seedlings and three or more independentlytransformed roots per construct were analyzed. Asshown by the representative images in Figure 3 andSupplemental Figure S2, all fusion proteins localized tothe NE in vivo. Additionally, some KASH proteins lo-calized to other cellular compartments, while MtWIT1could also be detected in the nucleoplasm (Fig. 3R).MtSINE1 was associated with fibrous structuresthat resemble actin cables (Fig. 3F), similar to the

Figure 2. Putative Medicago LINC complexcomponents localize to the NE in N. ben-thamiana leaf epidermal cells in a SIT-dependent manner. A (from top), Rows 1 and3: Localization of putative Medicago KASHproteins when coexpressed with H2B-RFP.Rows 2 and 4: Localization of putative Medi-cago KASH proteins with their SITs deleted. B,Localization of putativeMedicago KASH-bindingproteins SUNandWIT. C, Localization of rejectedputative Medicago LINC complex components.D, Quantification of NE localization using theNLIformula. Inset: NE, nuclear envelope maximumsignal; Cyt, cytoplasmic maximum signal. Datashown from at least 20 cells. Error bars = SE. Scalebars = 20 mm. ***Student’s t test, P value ,0.00001.






localization of Arabidopsis SINE1, which is known tocolocalize with actin in both N. benthamiana and Ara-bidopsis (Zhou et al., 2014). MtKASH5 localized to re-ticulate structures that resemble the endoplasmicreticulum (ER; Fig. 3P) and MtSINE5a was often foundin a punctate pattern of unknown nature at the nuclearrim (Fig. 3H). Together, these data indicate that theputative LINC complex components are robustly tar-geted to the NE in two dicot species, and that, at leastfor MtSINE1, the similar association with actin fibers inArabidopsis and Medicago might also indicate func-tional conservation. The expression pattern ofMtKASH5 is unknown andMtSINE5a is only expressedat a relatively low level in whole roots (SupplementalFig. S1). Although the localization data shown heremay therefore not fully represent endogenous locali-zation, there are examples ofMedicagoNE proteins thatare natively expressed at relatively low levels, are NE-localized when constitutively expressed, and play animportant role in symbiosis (Charpentier et al., 2016).

Diagnostic Protein–Protein Interactions Confirm MedicagoLINC Complexes

To determine if Medicago KASH and SUN proteinsform a complex, we performed coimmunoprecipitation

(Co-IP) experiments. To demonstrate that the interac-tions occur in a canonical fashion, we tested if the in-teractions depend on both a functional SIT in the KASHproteins and a functional SUN domain in the SUNprotein. Substitutions of two highly conserved residues(H439A, Y443F) within a region homologous to theKASH-binding lid of mammalian SUN2 were intro-duced into MtSUN to create MtSUNdmut. (Zhou et al.,2014). The equivalent mutations in the ArabidopsisSUN protein SUN2were shown to significantlyweakenSUN-KASH binding in Co-IP experiments (Zhou et al.,2014) and were also used here.GFP-tagged Medicago KASH proteins were coex-

pressed with Myc-tagged versions of either AtSUN2and AtSUN2dmut (Zhou et al., 2014) or MtSUN andMtSUNdmut in N. benthamiana via A. tumefaciens-mediated leaf infiltrations. To also test WIP/WITbinding, MtWIP1a and MtWIP1b were coexpressedwith Myc-tagged MtWIT1. Co-IP was performed withanti-GFP (Abcam), and proteins were detected withanti-GFP to detect the KASH protein (Roche or Clon-tech) or anti-Myc (Sigma) to detect SUN or WIT. Asshown in Figure 4, all Medicago KASH proteins thatlocalize to the NE, as well as ArabidopsisWIP1, interactwith both Arabidopsis SUN2 (Fig. 4A) and MedicagoSUN (Fig. 4B). This suggests that Medicago KASH andSUN proteins are capable of forming LINC complexes

Figure 3. PutativeMedicago LINC complex components are localized to the NE inMedicago transgenic hairy roots. (Left image,each column) Undifferentiated root tip cells. (Right image, each column) Differentiated cells, highlighting additional elements ofsome proteins’ localization (see, in particular, F and P). Representative images from at least three independently transformedroots. Scale bars = 20 mm.







Figure 4. PutativeMedicago KASH proteins interact with both Arabidopsis SUN2 andMedicago SUN in a SIT- and SUN domain-dependent manner. GFP-tagged KASH and 6xMyc-tagged SUN and WIT proteins were transiently expressed in N. benthamianaleaves and whole protein extracts were isolated and immunoprecipitated with an anti-GFP antibody. A to C, GFP-tagged KASHproteins were detected with anti-GFP, while 6xMyc-tagged SUN and WIT proteins were detected with anti-Myc. A, Co-IP ofMedicagoKASHproteins and Arabidopsis SUN2. B, Co-IPofMedicagoKASHproteins andMedicago SUN. C, Co-IPofMtWIP1a,MtWIP1b, the TMD fragment of Arabidopsis WIP1, and untagged GFP (free GFP) with MtWIT1. Asterisk: background band.Input: immunoprecipitated ratio = 1:9. Some proteins were expressed at too low a level to be detected in the input, but the





in planta, and that both SUN and KASH proteins inMedicago and Arabidopsis are capable of cross-speciesinteraction. Additionally, MtWIP1a and MtWIP1b in-teract with MtWIT1 (Fig. 4C), suggesting that thisprotein–protein interaction is conserved from Arabi-dopsis to Medicago. Together, these data show that ourbioinformatic approaches to identify Medicago LINCcomplex components were largely successful, and thatMedicago contains a complement of LINC complexessimilar to but exceeding that of Arabidopsis.

Medicago SINE1 Colocalizes with Actin and Alters ERMorphology in N. benthamiana

Arabidopsis SINE1 is known to colocalize with actinin both N. benthamiana leaf epidermal cells and Arabi-dopsis root cells (Zhou et al., 2014). Strikingly,MtSINE1and MtSINE1ΔSIT localize to fibrous structures in N.benthamiana, and MtSINE1 localizes to similar struc-tures in Medicago transgenic hairy roots (Figs. 2A and5A). To determine if these structures are cytoskeletalcomponents, we performed colocalization experimentswith MtSINE1 and several subcellular markers in N.benthamiana. Because MtSINE1ΔSIT is not retained atthe NE, this protein accumulates at the endoplasmicreticulum (ER) to a higher degree, facilitating visuali-zation of cytoplasmic interactions.GFP-MtSINE1 and GFP-MtSINE1ΔSIT were each

coexpressed in N. benthamiana leaf epidermal cells withone of four mCherry-tagged markers, marking the nu-cleus (Histone 2B), actin (Lifeact; Riedl et al., 2008),MTs, MT-associated protein 4 (MAP4), or the ER (Cal-nexin). As seen in Figure 5B, MtSINE1 and MtSI-NE1ΔSIT colocalize with Lifeact at cortical fibers.However, neither colocalizes with MAP4 (Fig. 5B). In-terestingly, when MtSINE1, and particularly MtSI-NE1ΔSIT, are coexpressed with the ER markerCalnexin, they also colocalize and appear to alter ERmorphology, making the normally reticulate pattern ofCalnexin (Fig. 5C) appear more actin-like (Fig. 5B).Together, these data indicate that MtSINE1 colocalizeswith the actin cytoskeleton, suggesting that MtSINE1might interact with actin in a direct or indirect manner,and that it may function in NE or ER attachment to theactin cytoskeleton.

A Dominant-Negative Approach To Deplete KASHProteins from the NE

In Arabidopsis, it is known that LINC complexesaffect nuclear morphology (Oda and Fukuda, 2011;Zhou et al., 2012a; Tamura et al., 2013) and nuclearmobility (Tamura et al., 2013; Zhou and Meier, 2014).

To probe the role of Medicago LINC complexes in thesephenomena, we utilized a dominant-negative fragmentof Arabidopsis SUN2, ERS-GFP-SUN2Lm (here called“SUNDN” for “SUN Dominant Negative”; Zhou et al.,2015b). SUNDN is composed of an N-terminal ER tar-geting signal, fluorophore (here eGFP or mRFP), thelumenal domain of Arabidopsis SUN2 including thecoiled-coil and SUN domains, and a C-terminal ER re-tention signal (HDEL; Fig. 6A). SUNDN has previouslybeen used inArabidopsis pollen to delocalize the KASHprotein AtWIP1 and its binding partner AtWIT1 fromthe vegetative NE, thereby causing vegetative nuclearmovement defects similar to a wip/wit null mutant(Zhou et al., 2015b).To determine whether SUNDN can similarly delo-

calizeMedicagoKASHproteins from theNE, we createda 35S-driven, red fluorescent protein (RFP)–taggedversion of SUNDN (RFP-SUNDN). We coexpressedRFP-SUNDN in N. benthamiana with GFP-tagged Med-icago LINC complex components via leaf infiltration(Fig. 6B, Supplemental Fig. S3). When coexpressedwithRFP-SUNDN, Medicago KASH and WIT proteins werestatistically significantly less enriched at the NE thanwhen they were coexpressed with RFP-tagged Histone2B (H2B-RFP; Fig. 6C; Student’s t-test P , 0.0005;Supplemental Fig. S4, Student’s t-test P , 0.05). Con-trastingly, MtSUN was not significantly depletedfrom the NE when coexpressed with SUNDN (Fig. 6C;Student’s t-test P . 0.1; Supplemental Fig. S4). Thissuggests that SUNDN is capable of binding to and de-pleting Medicago KASH proteins from the NE. This isin agreement with our previous observation that allMedicago KASH proteins are able to bind ArabidopsisSUN2 in a co-IP assay, as SUNDN is derived fromAtSUN2.To determine whether SUNDN can delocalize KASH

proteins from the NE in vivo, we cotransformed Medi-cago hairy roots with GFP-tagged MtWIP1b, MtSINE1,MtKASH4, or MtSUN and either RFP-tagged SUNDNor untagged RFP from the pH7WGR2 vector. Consis-tent with the data from N. benthamiana, we find thatGFP-tagged MtWIP1b, MtSINE1, and MtKASH4 arestrongly depleted from the NE when coexpressed withRFP-SUNDN, whereas NE enrichment of MtSUN onlymarginally changes (Fig. 6, D and E).

Root Hair Nuclear Morphology and Nuclear Mobility AreAltered upon Depletion of LINC Complexes at the NE inMedicago Hairy Roots

After confirmation that SUNDN depletes KASHproteins from the NE, we asked whether KASH proteindelocalization in SUNDN-expressing hairy roots altersnuclear morphology and/or mobility. For these assays,

Figure 4. (Continued.)immunoprecipitate shows that they are expressed. WT, wild type; dmut, double mutation; AtWIP1 TDF, the TMD fragment ofArabidopsis WIP1; IN, input; IP, immunoprecipitated.








we transformed Medicago hairy roots with either 35S-driven, eGFP-tagged SUNDN (SUNDN) or 35S-driven,untagged GFP from the pK7WGF2 vector. Whenexpressed in Medicago hairy roots, SUNDN localizes tothe NE and ER-like structures in undifferentiated cells,epidermal cells, and root hairs (SUNDN; Fig. 7A),whereas untagged GFP is diffuse throughout the nu-cleus and cytoplasm (free GFP; Fig. 7A). Due to theirrelevance to the early stages of rhizobial infection, wechose to analyze the cellular effects of SUNDN in ma-ture root hairs. To determine the degree of root hairnuclear circularity in SUNDN- and GFP-expressingcells, we calculated the circularity index (4[area/perimeter2]; Fig. 7B), which equals one for a disk. Thecircularity index for root hair nuclei in hairy rootsexpressing free GFP was 0.68 on average, indicative ofthe overall elongated shape of root hair nuclei. How-ever, root hairs expressing SUNDN had on averagemore circular nuclei (circularity index 0.82; Student’st test P , 0.00001; Fig. 7B), consistent with previouslypublished Arabidopsis data showing that LINC com-plex disruptions lead to increased circularity in roothair nuclei (Oda and Fukuda, 2011; Zhou et al., 2012a,2015a; Tamura et al., 2013).

To assess whether SUNDN influences nuclear mo-bility in root hairs, a minimum of 50 root hair nucleifrom SUNDN- and GFP-expressing Medicago hairyroots were tracked over a 60-min imaging period(50 z-stacks of 50 vertical sections each, 1.2 min apart).To facilitate initial qualitative observation, thesez-stacks were converted into maximal projections, asshown in Supplemental Movies S1 and S2 and therepresentative kymographs in Figure 7C. We observedthat SUNDN-expressing nuclei often moved rapidly, inmultiple directions, over the imaging period, whereasGFP-expressing nuclei moved relatively slowly overthe imaging period.

To quantify these phenomena, nuclei were tracked bydetermining the absolute distance in micrometers be-tween the center of the nucleus from frame n to frame n+1, generating a measure of nuclear displacement be-tween frames (frame-to-frame displacement). To tracknuclear movement in a manner comparable to Tamuraet al. (2013), these frame-to-frame displacements weresummed over the 50 frames (“Total displacement”;Fig. 7D). Based on this metric, SUNDN-expressingMedicago root hair nuclei, on average, travel a statisti-cally significantly greater total distance than GFP-expressing nuclei over the imaging period (analysis ofvariance [ANOVA] P , 0.02).

Figure 5. Medicago SINE1 colocalizes with actin, and impacts ERmorphology in N. benthamiana leaf epidermal cells. A, Localizationpattern of GFP-MtSINE1 (left) and GFP-MtSINE1ΔSIT (right) whencoexpressed with the nuclear marker RFP-Histone 2B. B (from top),Rows 1 and 4: GFP-MtSINE1 and GFP-MtSINE1Δ SIT coexpressed with

MT marker RFP-MAP4; rows 2 and 5: GFP-MtSINE1 and GFP-MtSINE1ΔSIT coexpressed with actin marker RFP-Lifeact (Riedl et al.,2008); rows 3 and 6: GFP-MtSINE1 andGFP-MtSINE1ΔSIT coexpressedwith ER marker RFP-Calnexin. (Left) Merge; (center) GFP; (right) RFP. C,Localization of RFP-tagged marker proteins in the absence of MtSINE1.Calnexin marks ER sheets and cisternae, Lifeact marks actin filaments,and MAP4 marks MTs. Scale bars = 20 mm.







To better capture the differences between SUNDN-and free GFP-expressing nuclei, we individually plot-ted all frame-to-frame displacements of every nucleus(“Frame-to-frame displacement”; Supplemental Fig.S5). We observed that, for the majority of the time, bothSUNDN- and free GFP-expressing nuclei displaced,2.8 mm from frame to frame. However, SUNDN-expressing nuclei experienced a greater range of frame-to-frame displacements than GFP-expressing nuclei, witha striking increase in frame-to-frame displacements above5 mm (Supplemental Fig. S5). To better capture this phe-nomenon, themaximum frame-to-framedisplacement foreach nucleus was identified and plotted (“Maximumframe-to-frame displacement”; Fig. 7E). The difference

between SUNDN- and GFP-expressing nuclei becomesmore apparent in this context, with SUNDN-expressingnuclei displaying on average greater maximum dis-placements than free GFP-expressing nuclei (ANOVAP , 0.0005). Together, these data suggest that SUNDN-expressing nuclei undergo, more frequently, rapid andlonger-distance movements than free GFP-expressingnuclei.To determine if the increased circularity of SUNDN-

expressing root hair nuclei is related to their increasedmobility, we plotted maximum frame-to-frame dis-placement against circularity. For SUNDN-expressingnuclei, circularity and maximum frame-to-frame dis-placement have a positive relationship, where the more

Figure 6. SUNDNdepletesMedicago KASH, but not SUN, proteins from the NE inN. benthamiana andMedicago. A, Graphicalrepresentation of the SUNDN construct adapted from Zhou et al. (2015b). B (top row), Representative images of localization ofGFP-tagged Medicago KASH and SUN proteins when coexpressed with H2B-RFP; (bottom row) representative images of lo-calization of GFP-tagged Medicago KASH and SUN proteins when coexpressed with RFP-SUNDN. C, Quantification of NElocalization using theNLI formula; data shown from at least 19 cells each. D, Representative images of localization of GFP-taggedMedicago KASH and SUN proteins inMedicago hairy roots when coexpressedwith RFP from the pH7WGR2 vector (free RFP) orRFP-SUNDN. E, Quantification of NE localization using the NLI formula. Data shown from at least 11 cells each. Scalebars = 20 mm. ***Student’s t test, P, 0.00005; *Student’s t-test, P, 0.025; ns, no significance with Student’s t test, P. 0.1. ERS,ER targeting signal; shown in blue color, unknown domain; CC, coiled-coil. Inset: NE, nuclear envelope maximum signal; Cyt,cytoplasmic maximum signal.








circular nuclei have higher maximum frame-to-framedisplacements, and the two variables are statisticallycorrelated (Fig. 7F, Pearson’s R, P , 0.0005). By con-trast, root hair nuclear circularity has no statisticallysignificant correlation to maximum frame-to-framedisplacement in GFP-expressing roots (Fig. 7F, Pear-son’s R, P = 0.15). RFP-SUNDN expression had sim-ilar cellular effects to expression of GFP-taggedSUNDN, including increases in both nuclear circu-larity and maximum frame-to-frame nuclear dis-placement (Supplemental Fig. S6). Taken together,these data suggest that both nuclear morphology andnuclear movement have been altered in SUNDN-expressing root hairs, and that more circular nucleiare more likely to be rapidly displaced from theirlocation.

SUNDN Expression Leads To Decreased IT and NoduleFormation in Medicago Hairy Roots

To determine whether the subcellular effects on nu-clear movement and shape have an impact on rhizobialsymbiosis, Medicago roots expressing GFP or SUNDNwere inoculated with Sinorhizobium meliloti strain 2011constitutively expressing dsRed (Sm2011-dsRed). Fourweeks after inoculation, SUNDN-expressing roots hadsignificantly fewer nodules relative to GFP-expressingroots (Fig. 8A, Student’s t-test P value , 0.001). How-ever, the nodules formed were fully developed andinfected (Fig. 8C). To assess the infection process,the number of IT and infection pockets were quanti-fied 10 d after inoculation with Sm2011 expressingb-galactosidase. Consistent with the reduction in nodule

Figure 7. SUNDN expression leads to more circular andmobileMedicago hairy root hair nuclei. A, Localization of SUNDN andfree GFP in Medicago hairy roots. Representative images from $6 independently transformed hairy roots. B, Circularity of roothair nuclei from SUNDN- and free GFP-expressing Medicago hairy roots. (Left) Scatter plot showing each nucleus as a singlepoint. (Right) Box plot. (Top line)Maximum. (Box)Quartiles. (Solidmiddle line)Median. (Dottedmiddle line)Mean. (Bottom line)Bottom fence. ***ANOVA, P value , ,0.00001. C, Kymographs of nuclear movement in GFP- and SUNDN-expressing roothairs. The x axis represents space, and the y axis represents time. Scale bars = 20 mm. D, Distance traveled by root hair nuclei ofSUNDN- and free GFP-expressing Medicago hairy roots. Total displacement (sum of all frame-to-frame displacements over 50frames, 1.2 min apart) is shown. (Left) Scatter plot showing each nucleus as a single point. (Right) Box plot. (Top line) Maximum.(Box) Quartiles. (Solid middle line) Median. (Dotted middle line) Mean. (Bottom line) Bottom fence.*ANOVA, P value, 0.02. E,Maximum frame-to-frame displacement of root hair nuclei of SUNDN- and free GFP-expressing Medicago hairy roots. (Left)Scatter plot showing each nucleus’ maximum frame-to-frame displacement as a single point. (Right) Box plot. (Top line) Topfence. (Box) Quartiles. (Solid middle line) Median. (Dotted middle line) Mean. (Bottom line) Minimum. ***ANOVA, P, 0.0005.F, Maximum frame-to-frame displacement as a function of the circularity of hairy root hair nuclei in SUNDN- and free GFP-expressing roots. (Blue points) Free GFP. (Orange points) SUNDN.






number, significantly fewer ITs and infection pocketswere seen on SUNDN-expressing roots (Fig. 8B, Student’st-test P value , 0.001; Supplemental Figure S7, Student’s

t-test P value, 0.01). However, the ITs that formed werewild-type–like, that is, the IT progressed normallythrough the epidermal cell layer to reach the cortex, andno deformation of the IT structure was observed (Fig. 8D,ITs shown in blue). These data suggest that SUNDNnegatively impacts IT initiation, which in turn leads to adecrease in nodule number.

DISCUSSION

KASH Proteins Outside of the Brassicaceae

In this study, we identifiedMedicago homologs of plantKASH proteins previously discovered in Arabidopsis,and also described several previously unknown, butwidely conserved, plant KASHproteins that are excludedfrom the Brassicaceae. Thismay indicate that the plantNEproteome is more diverse than previously thought, andthat mining additional proteomes, especially those out-side the Rosid clade, for KASH proteins will likely yieldfurther candidates. Additionally, several Arabidopsisgene families with two or three members (WIP1/2/3,WIT1/2, SUN1/2, SINE1/2) exist as a single copy in theMedicago genome, making this model legume a powerfulnew system to address LINC complex function in cell anddevelopmental biology as well as symbiosis.MtWIP1a and MtWIP1b interact with both MtSUN and

MtWIT1, suggesting that the SUN-WIP-WIT complex isconserved in Medicago, and that both MtWIP1a andMtWIP1b can participate in this complex. Similar to Ara-bidopsis AtSINE1, MtSINE1 and MtSINE1ΔSIT decorateactin filaments, with MtSINE1 and MtSINE1ΔSIT alsoappearing to alter ER morphology when overexpressed(Fig. 5). These data are consistent with the assumption thatMtSINE1 is associatedwith bothF-actin and theERandNEmembranes. This is reminiscent of the Arabidopsis SolubleN-ethylmaleimide-sensitive-factor Attachment Protein Re-ceptor protein SYP73, which directly connects the ERmembrane to the actin cytoskeleton (Cao et al., 2016). SYP73overexpression in tobacco (N. tabacum) leaf epidermal cellsleads to reorganization of the ER from a reticulate patternto an actin-like pattern, similar to MtSINE1ΔSIT over-expression. Further, this effect was expression-dependent,with low-expressing cells showing a combined reticulateand fibrous ER structure, similar to the pattern seen whenMtSINE1,which is also less abundant at the cortical ER thanMtSINE1ΔSIT, is overexpressed. We thus propose thatin vivo, NE-retained MtSINE1 acts to connect the NE andF-actin at the nuclear periphery. By contrast, recombinantMtSINE1ΔSIT is released from SUN interaction and is thusmore broadly associated with the cortical ER, where addi-tional ER-actin connections may cause the observed ERreorganization and closer ER-actin association.

SUNDN Expression Affects Nuclear Elongation andControlled Nuclear Movement in Medicago Root Hairs

Two effects on root hair nuclei were observed afterexpressing SUNDN inMedicago roots. First, nuclei were

Figure 8. SUNDN-expressing roots develop fewer nodules and ITs thanGFP-expressing roots. A, Quantification of nodule frequencies in freeGFP- and SUNDN-expressing Medicago hairy roots. Mean values andSEs shown from three biological replicates. Number of plants analyzedindicated on graph. B, Quantification of IT frequencies in GFP- andSUNDN-expressing Medicago hairy roots. Mean values and SEs shownfrom one biological replicate. Number of plants analyzed indicatedbelow graph. C, Representative nodules from experiment in (A). D,Representative ITs from the experiment in (B) are shown (visualized byblue 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside staining of thebacteria). ***Student’s t test, P , 0.001.






on average less elongated, consistent with prior studiesfinding that Arabidopsis loss-of-function mutations inWIP1, WIP2, and WIP3; WIT1 and WIT2; or SUN1 andSUN2 all cause a loss of root hair nuclear elongation (OdaandFukuda, 2011; Tamura et al., 2013;Zhou et al., 2015a).Second, root hair nuclear mobility was affected, suchthat although wild-type root hair nuclei showed littleoverall mobility during the 60-min imaging periods,SUNDN nuclei underwent sporadic, higher-velocity“spurts,” interrupted by periods of low mobility(Fig. 7; Supplemental Movies S1 and S2).

In Arabidopsis, it has been reported that a LINC com-plex consisting of SUN,WIP, WIT, and the motor proteinMYOSINXI-I contributes to nuclearmovement inmatureroot hairs and mutants in either MYOSIN XI-I or WIT1and WIT2 lead to reduced nuclear mobility (Oda andFukuda, 2011; Zhou et al., 2012a; Tamura et al., 2013).In Arabidopsis, no difference in root hair nuclearmobilitywas found in a sun1 sun2 double mutant, although nucleiexhibited a more circular morphology (Oda and Fukuda,2011). However, the double mutant tested consisted of anull T-DNA insertion in AtSUN1 (sun1-1) and a microRNA against AtSUN2 (sun2-KD), and it is thus possiblethat a residual amount of SUN2 anchors sufficient WIP/WIT/MyoXI-I complex to the NE to allow for wild-typelevels of movement to occur. In addition, nuclear move-ment in this study was assessed in developing root hairs,not in mature root hairs as in Tamura et al. (2013) andour study.

Here, we used the dominant-negative strategy thathad been successfully applied to Arabidopsis SUN2, aswell as animal SUN proteins (Crisp et al., 2006). InArabidopsis, SUNDN was expressed under a pollen-specific promoter and this expression faithfully phe-nocopied the effect of wip and wit loss-of-functionmutants on nuclear movement in pollen tubes, and wasshown to deplete WIP1 and WIT1 protein from the NEof the vegetative nucleus in pollen grains (Zhou et al.,2015b). Here, we found that expression of the sameSUNDN protein leads to a reduction of elongation inMedicago root hair nuclei (Fig. 7B). This is the samephenotype observed for loss-of-function mutants ofwip, wit, myosin xi-i, and sun in Arabidopsis (Oda andFukuda, 2011; Tamura et al., 2013; Zhou et al., 2015a),thus strongly suggesting that the SUNDN constructused here indeed affects the function of one or moreLINC complexes in Medicago root hairs.

In addition, we show that root hair nuclei in SUNDN-expressing Medicago roots can move more rapidly, andthat their increased circularity correlates with increasedmobility (Fig. 7, E and F). Importantly, our assay differsfrom previous studies in that SUNDN could potentiallydeplete a number of functionally distinct LINC com-plexes from the NE, not only SUN-WIP-WIT-MyoXI-i(Tamura et al., 2013). Therefore, the most plausible ex-planation for the observed release from wild-type po-sitioning seen here is that, in Medicago, certain LINCcomplexes are involved in restraining root hair nucleiagainst rapid movement, and that such connections aredisrupted by SUNDN expression.

Nuclei are thought to be both moved by and re-strained against cytoplasmic streaming in Drosophila(Baker et al., 1993; von Dassow and Schubiger, 1994;Bernard et al., 2018). In this system, it has been shownthat passive, cytoplasmic streaming-dependent nuclearmigrations are roughly three times faster than active,MT-dependent nuclear migration, with cytoplasmicstreaming movements averaging 23.2 mm/min andMT-dependent nuclear migration averaging 7.68 mm/min (Baker et al., 1993). Cytoplasmic streaming in theshank of Medicago root hairs has been shown to reachspeeds between 8 and 14 mm/s (Sieberer and Emons,2000). In this cell type, SUNDN-expressing nuclei ex-hibit a maximum velocity of 13.89 mm/min (16.67 mm/1.2 min), and free GFP-expressing nuclei exhibit a maxi-mum velocity of 4.45 mm/min (5.35 mm/1.2 min; Fig. 7).These data mirror those from Drosophila, with SUNDN-expressing nuclei exhibiting amaximumvelocity roughlythree times that of free GFP-expressing nuclei. This mayindicate that the relatively rapid nuclear movements inSUNDN-expressing root hairs are cytoplasmic streaming-dependent, as compared to the slower, LINC complex-dependent movements in free GFP-expressing roothairs. Taken together, these data suggest that LINCcomplexes may function in plants to both move nucleiand restrain them against cytoplasmic streaming, as adual nuclear movement/retention system.

SUNDN as a Tool to Study LINC Complexes acrossPlant Species

Previously, SUNDN had been used to delocalizeAtWIP1 and its ONM-interacting partner AtWIT1 inthe context of a SUN knockdown mutant (sun1-KO/sun2-KD) in Arabidopsis pollen (Zhou et al., 2015b).Here, we show that SUNDN expression decreases bothAtWIP1 and Medicago KASH protein enrichment at theNE in both N. benthamiana and Medicago. All testedMedicago KASH proteins were depleted from the NE,suggesting that SUNDN has the potential to interferewith the entire KASH proteome of a plant. The fact thatnuclear shape andmovement are changed in aMedicagowild-type context suggests that LINC complex functioncan be broadly inhibited without mutating endogenousSUN-encoding genes. Previously, a broad disruption ofLINC complexes has been prevented by the meioticlethality of SUN null mutants (Varas et al., 2015). Al-though SUN knockdown mutants exist in plants, theyare not strong enough to phenocopy KASH null mu-tants in all cases (Oda and Fukuda, 2011; Varas et al.,2015; Zhou et al., 2015b).

Although it would be difficult to quantify the degreeof LINC complex disruption in SUNDN-expressingcells, we appear to have efficiently outcompeted na-tive SUN proteins for SIT domain binding based on thesimilarity of the nuclear shape phenotype we observedhere to previously published data in KASH mutants(Fig. 7; Tamura et al., 2013; Zhou et al., 2015a). How-ever, some SUNDN and KASH protein remains at the






NE (Fig. 7), and it is therefore possible that KASHprotein functions requiring minimal NE localizationmay be retained in SUNDN-expressing cells.SUNDN only minimally alters SUN localization,

suggesting that it acts by outcompeting native SUNproteins for SIT domains, rather than targeting nativeSUNs to the ER in SUN-SUNDN complexes. This in turnsuggests that the inner nuclear membrane proteome re-mains unperturbed, thus likely focusing the disruptionon the interactions of LINC complexes with cytoplasmicpartners or other ONM proteins. Together, this makesSUNDN a valuable tool for exploring LINC complexfunction in plant species amenable to transformation, butwithout robust directed mutagenesis tools.

SUNDN Expression Affects IT and Nodule Formation

Given the reported correlations between nuclear posi-tioning and IT formation, factors involved in root hairnuclear mobility andmorphology are candidates for newplayers in the nodulation pathway.We find that SUNDNexpression indeed leads to defects in nuclearmorphology/mobility and a reduction in both IT and nodule forma-tion (Figs. 7 and 8). A working hypothesis to be tested inthe future is that SUNDN uncouples the root hair nucleusfrom nodulation-factor signaling, which leads to nuclearrepositioning, either by a change in nuclear position or by acurrently unknown mechanism. Further experiments di-rectly characterizing how nuclei move during IT progres-sion and how nuclear signaling events are affected inSUNDN-expressing roots will be necessary to test thishypothesis. In addition, this study now provides the toolsto address which of the Medicago KASH protein(s) aremost important for both nuclear retention and symbiosisinitiation, and to test if and how these two events arefunctionally connected.

CONCLUSION

Our study reveals the diverse LINC complexes encodedby theMedicago genome and explores their roles in nucleardynamics and symbiosis. The characterization ofMedicagoLINC complex components indicates that LINC com-plexes are conserved across plant species, and thatSUN-KASH interactions canoccurbothwithin andbetweenspecies. Expression of SUNDN inMedicago transgenic hairyroots reveals that LINC complex functionality is also con-served across species. Further, we provide the first evi-dence that LINC complexes, and possibly their role innuclear shaping andmovement, are involved in the earlieststages of symbiosis betweenMedicago and rhizobia.

MATERIALS AND METHODS

Candidate Gene Identification

Tobegin the in silico analyses, aBLASTsearch to identifyhomologsof knownArabidopsis (Arabidopsis thaliana) KASH and KASH-interacting proteins was

performed. BLASTp was used to search the nr/nt database, with a cutoff of 1e-05. Using these means, two homologs of AtWIP1 (MtWIP1a/Mt8g070940,MtWIP1b/Mt3g009740), two AtWIT1 homologs (MtWIT1/Mt7g017690,MtWIT2/Mt3g086660), a homolog of AtSINE1 (MtSINE1/Mt6g032885), and ahomolog of AtSUN1 (Mt8g043510) were identified.

The DORY algorithm has previously been shown to identify novel KASHproteins originating fromspecies across eukaryotes (Zhou et al., 2014), includingMedicago truncatula SINE5 (Mt2g033900; Zhou et al., 2014). Due to the presenceof a putative paralog of this gene, SINE5 was renamed MtSINE5a, and itsparalog Mt5g054260 SINE5b. For the DORY analysis in this study, theMedicagoproteome 4.0 v1 was downloaded from theMedicago genome database (http://www.medicagogenome.org/downloads) and subjected to the DORY algo-rithm. DORYwas set to a TMD frame length of 20, hydrophobic threshold of 40,maximum KASH tail length of 40, minimum tail length of 4, and protein lengthrange of 100–100,000 amino acids. The KASH tail was defined as [DTVAMPLIFY][VAIPL]PT. This led to the identification of MtKASH1/Mt3g099060,MtKASH4/Mt4g036225, MtKASH5/Mt6g016290, and MtKASH6/Mt1g052620.All putative KASHproteins that were originally identified using BLAST (MtWIP1a,MtWIP1b, and MtSINE1), as well as the paralogs SINE5a and SINE5b, were againidentified during this DORY search.

Cloning and Plasmid Construction

Medicago seedling cDNA was prepared by extracting RNA from 7-d-oldMedicago seedlings (RNeasy Kit, Qiagen 74104) and subjected to reversetranscription-PCR using random hexamer primers (Superscript III Kit, Invi-trogen 18080093). The ORFs ofMedicago LINC complex components were PCR-amplified with a high-fidelity polymerase (Phusion, NEB M0530S) from eitherthe cDNA or commercially synthesized pUC57 clones (Genscript). PCR primerswere designed to amplify the ORFs as annotated in version 4.0 of the Medicagogenome database (http://www.medicagogenome.org/). For cloning primers,see Supplemental Table S3. In addition to the cDNA-specific sequences, com-plementarity sequences designed for Gateway cloning into pDONR221 orTOPO reaction into pENTR/D-TOPO (Invitrogen; Supplemental Table S1)were also added to the primers. Sequences amplified with Gateway primerswere cloned into the pDONR221 vector using BP clonase (Thermo Fisher Sci-entific 11789020). Genes amplified with TOPO primers were cloned intopENTR/D-TOPO using the TOPO Cloning Kit (Invitrogen 45-0218). Plasmidswere isolated from the resulting colonies and the vector sequences wereconfirmed.

In a second round of PCR cloning, shortened fragments with the terminal 12nucleotides before the stop codon deletedwere cloned to generate ΔSIT versionsof the KASH proteins. New reverse primers were designed to anneal 59 of these12 nucleotides, and to replace the gene’s endogenous stop codon sequence.Using these and TOPO-compatible forward primers, ΔSIT clones were PCR-amplified and cloned into pENTR/D-TOPO as above, and the sequence wasconfirmed.

For localization studies in Nicotiana benthamiana and M. truncatula, cDNAsequences were moved from pENTR/D-TOPO to pK7WGF2 (Karimi et al.,2002) by LR reaction (Thermo Fisher 11791100) to obtain GFP-tagged ver-sions of the proteins (for example, GFP-MtWIP1a). For Co-IP, MtSUN andMtWIT1 were cloned into pGWB21 (Nakagawa et al., 2007) to obtain35S-driven, N-terminally 6xMyc-tagged versions of their proteins (e.g.Myc-MtSUN). MtSUNdmut was obtained by site-directed mutagenesis(QuikChange, Agilent 200521) using primers MtSUNdmut_sense andMtSUNdmut_anti-sense (Supplemental Table S3).

To create 35S-driven RFP-SUNDN, ERS-RFP-AtSUN2Lm-HDEL was am-plified from pK7WGRERS52 (Zhou et al., 2015b) via PCR and cloned intopENTR/D-TOPO (Life Technologies). After confirmation by sequencing, ERS-RFP-AtSUN2Lm-HDEL was cloned into the 35S-driven plant expressionvector pH7WG2 via LR reaction (Life Technologies) to create 35S:ERS-RFP-AtSUN2Lm-HDEL.

Transformation of Agrobacterium tumefaciens andAgrobacterium rhizogenes

A. tumefaciens strain GV3101 and A. rhizogenes strain AR1193 were trans-formed with plasmids described above by electroporation (Wise et al., 2006).Resulting GV3101 transformants were grown on solid lysogeny broth (LB)media with 20 mg/mL rifampicin and 30 mg/mL gentamycin, with 100 mg/mLspectinomycin to select for pK7WGF2-transformed strains and 50 mg/mLhygromycin to select for pGWB21-transformed strains. Resulting AR1193




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transformants were grown on solid LB media with 20 mg/mL rifampicin and100 mg/mL carbenicillin, with 100 mg/mL spectinomycin to select for binaryvectors.

Plant Materials and Growth

M. truncatula R108 seedlings were grown in upright plates on modifiedFåhraeus medium agar (0.9 mM CaCl2, 0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM

Na2HPO4, 20 mm Ferric Citrate, 0.5 mM NH4NO3, 100 mg/mL MnCl2, 100 mg/mL CuSO4, 100 mg/mL ZnCl2, 100 mg/mL H3BO3, 100 mg/mL Na2MoO4, and1.5% w/v Ultrapure Agar) under long days (16-h d/8-h n) at 20°C in a plantincubator. Plants were grown in a 1:1 mixture of sharp sand (Quikrete) andRedi-Earth (Sungro) under long days at an average temperature of 22°C. N.benthamiana plantswere grown at 28°C in soil under constant light in Redi-Earth(Sungro).

N. benthamiana Transient Transformation

LINC complex component proteins and markers were coexpressed in N.benthamiana leaves by Agrobacterium infiltration as described in Sparkes et al.(2006). Briefly, cultures were grown overnight and centrifuged to collect thecells. They were resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES,pH 5.4, and 100 mM acetosyringone) toOD600 = 1, and then pressure-infiltratedinto N. benthamiana leaves using a plastic syringe. For imaging experiments,plants were grown for 2 d after infiltration. For Co-IP experiments, plants weregrown for 3 d to increase expression.

For imaging experiments, sections of infiltrated leaves were cut out andimaged using a confocal microscope (Nikon Eclipse C90i) with small ormediumpinhole, gain setting range of 6.0 to 8.0. The 488-nmand561-nm laserswere set to20%. All images were taken at room temperature (RT) using water as the me-dium with a Plan Apochromat VC 603 H lens (numerical aperture of 1.4;Nikon), and acquired using NIS-Elements AR software, version 3.2 (Nikon).The transmitted light detector was turned on to collect transmitted light signalsimultaneously. Images were exported to PNG format by the software NIS-Elements (Nikon) and organized in the softwares Photoshop and Illustrator(Adobe).

Nuclear Localization Index Calculation

Images of fluorescent protein-expressing N. benthamiana leaf epidermal cellsor Medicago root epidermal cells were taken at an optical section with maximalnuclear radius and cross-sectioned cytoplasm (i.e. cells with the nucleus at thetop or bottom of the cell were not imaged, as a cross section of vertical cyto-plasm could not be obtained). At least 20 of these images were obtained perinfiltration. From these images, fluorescence profiles, crossing both the NE andthe cortical GFP signal several times, were obtained by analysis with the soft-ware NIS-Elements (Nikon). From these profiles, the two highest NE fluores-cence values and the two highest cortical fluorescence valueswere identified foreach cell. The twoNEmaximawere summed and divided by the sum of the twocytoplasmic maxima to generate the nuclear localization index.

Co-IP

Co-IP experiments were performed as described in Zhou et al. (2014).Briefly, N. benthamiana leaves were fully infiltrated with Agrobacterium culturesat an OD600 = 1 and allowed to grow for 3 d (see above). GFP fluorescence wasconfirmed using a confocal microscope (Eclipse C90i; Nikon), and leaves weresubsequently collected and ground in liquid N to a fine powder. Protein ex-traction was performed in radioimmunoprecipitation assay (RIPA) buffer(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% w/v SDS, 0.5% w/v sodiumdeoxycholate, 1% v/vNP-40, 1 mM phenylmethylsulfonyl fluoride, and 1% (v/v)protease inhibitor cocktail; Sigma-Aldrich) by applying 1 mL RIPA buffer to500 mL frozen tissue. One-tenth of the protein extract was used as the inputsample, and the rest was used for immunoprecipitation using protein A–

Sepharose beads (GE Healthcare) precoated with a rabbit anti-GFP antibody(Abcam ab290). After three washes using RIPA buffer, the immunoprecipitateswere diluted 3:1 with SDS loading buffer and boiled for 3 min. The sampleswere separated by 7.5% (v/v) or 10% (v/v) SDS-PAGE, transferred to poly-vinylidene difluoride (Bio-Rad) or nitrocellulose membranes (Bio-Rad), and analyzedby immunoblotting with a mouse anti-GFP antibody (1:2000, Roche 11814460001 orClontech 632569) or a mouse anti-Myc antibody (1:2000; Sigma-Aldrich M5546).

Secondary detection was performed with a sheep anti-mouse antibody conju-gated with horseradish peroxidase (1:20,000 Sigma-Aldrich A5906) andSuperSignal West Femto Maximum Sensitivity chemiluminescent substrate(Thermo Fisher Scientific 34095).

Hairy Root Transformation

Hairy root transformation ofMedicago seedlings was performed similarly toChabaud et al. (2006). Seeds were scarified with sandpaper and sterilized with25% v/v bleach before being germinated on 0.8% w/v water agar plates. A.rhizogenes strain AR1193 transformed with plasmids encoding 35S-driven,N-terminally GFP-tagged LINC complex components, 35S-driven GFP- or RFP-SUNDN, or 35S-driven free GFP or RFP were incubated for 2 d on LB agar(1.5% w/v) plates with 100-mg/mL spectinomycin, 20-mg/mL rifampicin, and100-mg/mL carbenicillin. Seedling root tips were excised and Agrobacteriawere harvested from secondary plates by scraping and painted on the seedlingroot and hypocotyl. M. truncatula R108 seedlings were grown in upright plateson modified Fahraeus medium agar (1.5%w/v) under long days (16-h d/8-h n)at 20°C in a plant incubator. The samples were imaged using a confocal mi-croscope (Eclipse C90i; Nikon) with the 488-nm laser set to 20% and the gain setbetween 6.5 and 8.0. All images were taken using on-plate microscopy as de-scribed in Genre et al. (2005). Seedlings were placed on a ModFP-agar solidplate, immersed in water, and Lumox film (Sarstedt) was placed on top. Allroots were imaged using a 403water immersion objective (numerical apertureof 0.8; Nikon), and acquired using NIS-Elements AR version 3.2 (Nikon). Thetransmitted light detector was turned on to collect transmitted light signalsimultaneously. Images were exported to PNG format by NIS-Elements(Nikon) and organized in Photoshop and Illustrator (Adobe).

Circularity Index Calculations

Singly transformed roots expressing GFP-SUNDN, RFP-SUNDN, free GFP,or free RFP were immersed in water under Lumox film (Sarstedt) and imagedusing a confocal microscope (Nikon Eclipse C90i) with the 488-nm or 561-nmlaser set to 20% and the gain set between 7.5 and 8.05. Images were taken usinga 403 water immersion objective (numerical aperture of 0.8; Nikon), and ac-quired using NIS-Elements AR version 3.2. Six independently transformedroots were visualized for GFP-SUNDN and free GFP, while two independentlytransformed roots were visualized for RFP-SUNDN and free RFP. Twenty roothair nuclei per root were visualized by creating z-stacks of sufficient 0.5- to0.55-mm sections to capture the entire nucleus. Amaximum intensity projectionof each nucleus was generated, and the software FIJI/ImageJ (Schindelin et al.,2012) was used to calculate the area and perimeter of each nucleus. Circularityindices were calculated by dividing the area by the perimeter squared, andplotted in a box plot with the whiskers being the upper and lower fences, thebox being the first and third quartiles, the middle solid line being the median,and the middle dashed line being the mean.

Live Imaging

Singly transformed roots expressing GFP-SUNDN, RFP-SUNDN, free GFP,or free RFP were immersed in water under Lumox film (Sarstedt) and imagedusing a confocal microscope (Nikon Eclipse C90i) with the 488-nm or 561-nmlaser set to 20% and the gain set between 7.5 and 8.05. Six independentlytransformed roots were visualized for GFP-SUNDN and free GFP, while threeindependently transformed roots were visualized for RFP-SUNDN and freeRFP. Images were taken using a 403 water immersion objective (numericalaperture of 0.8; Nikon), and acquired using NIS-Elements AR version 3.2.Z-stacks of 50 3-mm sections were taken at 1.2-min intervals. For analysis,z-stacks were converted into maximum intensity projections and nuclei weretracked using the manual tracking function in the software ImageJ. Manualtracking generated frame-to-frame displacement in two dimensions, whichwasthen analyzed four ways. First, all individual frame-to-frame displacements foreach nucleus were plotted via box plot, with the whiskers being the upper andlower fences, the box being the first and third quartiles, the middle solid linebeing the median, and the middle dashed line being the mean (SupplementalFig. S5). Second, the total displacement of each nucleus was calculated and allthe totals were plotted similarly to the individual displacements (Fig. 7D).Third, maximum single frame-to-frame nuclear displacement was calculatedand plotted (Fig. 7E). Fourth, circularity indices for tracked nuclei were calcu-lated as above, and displacement was plotted relative to circularity (Fig. 7F).







Kymographs were generated in NIS-Elements AR version 3.2 by generating amaximum intensity projection of the z-stacks generated above and drawing aline over the longitudinal axis of the root hair (Fig. 7C).

Nodulation Assay

Hairy root transformations were analyzed by confocal microscopy. Untrans-formed or chimeric roots were removed, and composite plants were moved topresterilized nutrient-poor soil (50% v/v mason sand, Quikrete; 50% v/v clay,Turface). Plants were allowed to recover in soil for 2 d. Sinorhizobium meliloti strainSm2011-dsRedwas grown for 2 d on solid tryptone yeast agar (0.5%w/v tryptone,0.3% w/v yeast extract, 0.012 M CaCl2, and 1.5% w/v agar, pH 7.5) plates with50 mg/mL streptinomycin and 5 mg/mL tetracycline, followed by overnight culti-vation in 5-mL tryptone yeast medium with the same antibiotics at 30°C. Theovernight culture was diluted in sterile deionized water to an OD600 = 0.001 andapplied to the composite plants in soil (;200mLculture per liter of substrate). Plantswere grown for 3 to 4 weeks, at which point they were removed and roots werechecked for GFP expression. Those roots that were still expressing GFP-taggedproteins of interest with the correct subcellular localization were then imaged fornodule colonization and nodules per plant were counted. To generate images ofnodule colonization, nodules were sliced open and imaged using a confocal mi-croscope (Nikon Eclipse C90i) with the 488-nm laser set to 20%, small pinhole, gainsetting range of 6.0 to 8.0. All images were taken at RT using water as the mediumwith a Plan Fluor 43 lens (numerical aperture of 1.3; Nikon), and acquired usingNIS-Elements AR version 3.2 (Nikon). The transmitted light detector was turned onto collect the transmitted light signal simultaneously. Images were exported toportable network graphic format by NIS-Elements software (Nikon) and organizedin Photoshop and Illustrator (Adobe).

IT Assay

Hairy root transformations were analyzed by confocal microscopy. Un-transformed or chimeric rootswere removed, and composite plantsweremovedto 1.5%w/vwater agar plates with 100 nM aminoethoxyvinyl Gly covered withfilter paper (Whatman qualitative filter paper for technical use, grade 0858,grained, 110 3 580 mm; cat. no. WHA10334365, Sigma-Aldrich). Roots weresprayed with 1 mL of OD600 = 0.03. Sm2011 expressing LacZ using a MADSdevice was covered with an upper layer of filter paper; the plates were closedand the bottom-half of the plates was covered with black felt. Plants weregrown under long-day conditions (16-h d/8-h n) at 20°C in a plant incubator for10 d. Plants were then stained as in Journet et al. (1994). For ITs in Figure 8, hairyroots were removed from the apical portion of the plants and fixed in 1.25% (v/v)glutaraldehyde in Z9 buffer (100mMNaPO4, 10mMKCl, and 1mMMgSO4), firstsubjected to a vacuum for 15 to 30min, then incubated for 1 h at RT. Plants werethen rinsed three times for 5min each in Z9 buffer and stained for 3 to 4 h at 30°Cin the dark in 0.1 M sodium P (pH 7.2), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and0.02 M 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside. ITs and infectionpockets were visualized by the blue-stained bacteria and scored using a lightmicroscope (Zeiss Axiophot). After being rinsed three times for 10 min in Z9buffer and twice in distilled, sterile water for 5min, plants were placed on slidesand ITs and infection pockets were counted per centimeter root length.

For ITs in Supplemental Figure S7, hairy roots were removed from the apicalportion of the plants and fixed in 1.25% (v/v) glutaraldehyde in Z9 buffer (100 mM

NaPO4, 10mMKCl, and 1mMMgSO4) for 1 h at RTwith agitation. Plants were thenrinsed three times for 5min each in Z9 buffer and stained overnightwith 5mMK3Fe(CN)6, 5 mM K4Fe(CN)6, and 0.8 mg/mL 5-Bromo-6-chloro-3-indoxyl b-D-galacto-pyranoside Magenta b-D-galactoside in Z9 buffer. After being rinsed three times for10 min each in Z9 buffer and twice for 5 min each in sterile distilled water, plantswerewet-mounted on slides and ITswere visualized by purple-stained bacteria andscored using a dissecting microscope (Nikon SMZ1270).

Accession Numbers

Sequence data from this article can be found in the GenBank and NCBI datalibraries under the accession numbers provided in Supplemental Tables S1and S2.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Microarray expression of MtWIP1a, MtWIP1b,MtWIT1, MtWIT2, MtSINE5a, MtSINE1, and MtSUN in M. truncatulatissues.

Supplemental Figure S2. Medicago LINC complex components are local-ized to the NE in Medicago hairy root transformants.

Supplemental Figure S3. Coexpression with SUNDN depletes allMedicagoKASH proteins and ONM-KASH interactors, but not SUN, from the NEin N. benthamiana.

Supplemental Figure S4. Quantification of data in SupplementalFigure S3.

Supplemental Figure S5. SUNDN-expressing root hairs have a greaterrange of frame-to-frame nuclear displacements than GFP-expressingroot hairs.

Supplemental Figure S6. RFP-SUNDN has similar effects on nuclear shapeand movement as GFP-tagged SUNDN.

Supplemental Figure S7. SUNDN-expressing roots develop fewer ITs thanGFP-expressing roots.

Supplemental Table S1. Gene numbers, GenBank protein sequence acces-sion numbers, and corresponding Affymetrix chip probe numbers.

Supplemental Table S2. GenBank and NCBI reference sequence numbersfor proteins used in alignments shown in Figure 1, C and D.

Supplemental Table S3. Primers used for cloning.

Supplemental Movie S1. Nuclear movement in the root hairs of free GFP-expressing Medicago hairy roots.

Supplemental Movie S2. Nuclear movement in the root hairs of SUNDN-expressing Medicago hairy roots.

ACKNOWLEDGMENTS

A.H.N.-G. and I.M. thank Katherine Beigel for technical assistance and Dr.Xiao Zhou and Norman Groves for productive conversations. I.M. would liketo thankDr. Giles Oldroyd for hosting her during a sabbatical stay that gave riseto this project.

Received September 10, 2018; accepted December 2, 2018; published December10, 2018.

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